Distribution of carotenoids in sub-cellular and sub-plastidic fractions of radish seedlings (Raphanus sativus) grown in the presence of bleaching herbicides

The intracellular and intraplastidic distribution of carotenoids has been investigated in radish seedlings grown in the presence of the herbicides amitrole and SAN 6706. Both herbicides caused bleaching and the plants became deficient in chlorophylls and the usual chloroplast cyclic carotenoids, but accumulated the acyclic carotenoid biosynthetic intermediates 15-cis-phytoene and all-trans-lycopene. In both the untreated and herbicide-treated plants all carotenoids, including phytoene and lycopene, were contained in the plastid. In all cases the normal cyclic carotenoids were located virtually exclusively in the thylakoid or prothylakoid fraction. In amitrole-treated plants, lycopene also was contained only in the thylakoid fraction, whereas phytoene, in these and in SAN 6706-treated plants, was detected in both the thylakoid fraction and an envelope preparation. Possible implications for the biosynthesis of the carotenoids are discussed.     

The carotenoid pigments in the juice and flavedo of a mandarin hybrid (Citrus reticulata) cv michal during ripening

The carotenoid pigments ofthe mandarin hybrid (Citrus reticulata) cv Michal, in the juice and flavedo were characterized at three ripening stages, before, during and after colour break. During ripening the characteristic mandarin pattern was formed in the juice, which contained cryptoxanthin as the principal pigment. In the flavedo the chloroplast carotenoid pattern of the green fruit changed into the characteristic pattern of C. reticulata with a high level of citraurin which, together with cryptoxanthin, imparts an intensive reddish tint to the hybrid. The flavedo contained an unusual C₃₀ apocarotenoid, β-citraurinene (8′-apo-β-caroten-3-ol). The flavedo carotenoids of this accidental hybrid were compared with the carotenoids of the presumed parents Dancy tangerine and Clementine. The hybrid resembles more the second parent, from which it inherited the ability to biosynthesize a higher amount of citraurin as well as citraurinene. Citraurinene, considered a Citrus hybrid-specific pigment, was found for the first time in a Citrus variety. A possible biosynthetic pathway of the Citrus C₃₀ -apocarotenoids is proposed.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Regulation of UDP-Galactose:Ceramide Galactosyltransferase and UDP-Glucose:Ceramide Glucosyltransferase After Crush and Transection Nerve Injury

The enzyme activities of ceramide galactosyltransferase and ceramide glucosyltransferase were assayed as a function of time (0, 1, 2, 4, 7, 14, 21, 28, and 35 days) after crush injury or permanent transection of the adult rat sciatic nerve. These experimental models of neuropathy are characterized by the presence and absence of axonal regeneration and subsequent myelin assembly. Within the first 4 days after both injuries, a 50% reduction of ceramide galactosyltransferase-specific activity was observed compared to values found in the normal adult nerve. This activity remained unchanged at 7 days after injury; however, by 14 days the ceramide galactosyltransferase activity diverged in the two models. The activity increased in the crushed nerve and reached control values by 21 days, whereas a further decrease was observed in the transected nerve such that the activity was nearly immeasurable by 35 days. In contrast, the ceramide glucosyltransferase activity showed a rapid increase between 1 and 4 days, followed by a plateau that was 3.4-fold greater than that in the normal adult nerve, which persisted throughout the observation period in both the crush and transection models. [3H]Galactose precursor incorporation studies at 7, 14, 21, and 35 days after injury confirmed the previously observed shift in biosynthesis from the galactocerebrosides during myelin assembly in the crush model to the glucocerebrosides and oligohexosylceramide homologues in the absence of myelin assembly in the transection model. The transected nerves were characterized by a peak of biosynthesis of the glucocerebrosides at 14 days. Of particular interest is the biosynthesis of the glucocerebrosides and the oligohexosylceramides at 7 and 14 days after crush injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Decomposition of cellulose,soil organic matter and plant litter in a temperate grassland soil

The formation of mor humus in an experimental grassland plot, which has been acidified by long-term fertiliser treatment, has been studied by comparing the rates of cellulose, soil organic matter and plant litter decay with those in an adjacent plot with near-neutral pH and mull humus. The decomposition of cellulose filter paper in litter bags of 5 mm, 1-mm and 45-μm mesh size buried at 3 to 4 cm depth the plots was followed by measuring the weight loss and changes in glucose content over a 6 month period. Soil pH was either 5.3 or 4.3. Decomposition of native soil organic matter and plant litter in soil from the same plots were followed using CO₂ evolution in laboratory microcosms. Cellulose weight loss at pH 5.3 was greatest from the 5-mm mesh bags and least from the 45-um mesh bags. At pH 4.3 there was little weight loss from bags and no significant differences in weight loss between bags with different sized mesh. There was, however, a reduction in the glucose content of the hydrolysed and derivatised filter paper with time. The decomposition rate of native soil organic matter in the low pH soil was increased to that observed in the less acid soil when the pH of the former was increased from 4.3 to 5.3. The increase in decomposition rate of added plant litter in the more acid soil as a result of CA(OH)₂ addition was only 60% of that observed in the soil with pH 5.3. These data support the hypothesis that the absence of soil animals and the restricted microbial decomposition in the acidic soil was responsible for mor humus formation.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Use of Cellulose Acetate Electrophoresis in the Taxonomy of Steinernematids (Rhabditida,Nematoda)

A steinernematid nematode was isolated from soil samples collected near St. John''s, Newfoundland, Canada. On the basis of its morphometry and RFLPs in ribosomal DNA spacer, it was designated as a new strain, NF, of Steinernema feltiae. Cellulose acetate electrophoresis was used to separate isozymes of eight enzymes in infective juveniles of S. feltiae NF as well as four other isolates: S. feltiae Umeå strain, S. feltiae L1C strain, Steinernema carpocapsae All strain, and Steinernema riobravis TX strain. Based on comparisons of the relative electrophoretic mobilities (μ) of the isozymes, one of the eight enzymes (arginine kinase) yielded zymograms that were distinctive for each of the isolates, except for the Umeå and NF strains of S. feltiae, which had identical banding patterns. Four enzymes (fumarate hydratase, phosphoglucoisomerase, phosphoglucomutase, and 6-phosphogluconate dehydrogenase) yielded isozyme banding patterns that were characteristic for all isolates, except for the L1C and NF strains of S. feltiae, which were identical. Two enzymes (aspartate amino transferase and glycerol-3-phosphate dehydrogenase) yielded zymograms that permitted S. carpocapsae All strain to be discriminated from the other four isolates, while the remaining enzyme (mannose-6-phosphate isomerase) was discriminatory for S. riobravis TX strain. Except for one enzyme, the isozyme banding pattern of the NF isolate of S. feltiae was the same as in the L1C strain, isolated 13 years previously from Newfoundland. Cellulose acetate electrophoresis could prove invaluable for taxonomic identification of isolates of steinernematids, provided that a combination of enzymes is used.     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.